CRISPR-Cas9-Based Functional Analysis of Immune Evasion Mechanisms and Vaccine Candidate Identification in Leishmania

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Background: Leishmaniasis continues to be a major health problem worldwide because the parasite has developed several strategies to resist the immune response, making the development of vaccines and treatment challenging.Methods: In the present work, we have used the CRISPR-Cas9 gene-editing system to explore the mechanisms of immune evasion used by Leishmania, and to identify new vaccine targets.Important immune-regulation genes, such as LACK, GP63, and CPB, have been successfully deleted in Leishmania major and Leishmania donovani using PCR and Sanger sequencing.Results: The immunological profile of gene-deleted BALB/c mice was assessed following infection.The modified parasites caused a greater infiltration of CD8+ IFN-γ+ T cells than did wild-type parasites.Cytokine protein levels showed elevated IFN-γ and TNF-α, and decreased IL-4, indicating a highly polarized Th1-type immune response.Immunization with genetically engineered antigens was used to assess vaccine efficacy.Immunization with antigens from the deleted parasites resulted in a significant rise in IgG antibody levels, the IgG1/IgG2a ratio, lymphocyte proliferation, and cytotoxic T cell activity.More importantly, survival rates of 80% in vaccinated mice were significantly higher after challenge with wildtype Leishmania than in 10% of mice immunized with wild-type antigens. Conclusions:The results demonstrate that host-protective immunity can be boosted by inactivating genes that contribute to immune evasion using the CRISPR-Cas9 technique.These approaches yield promising antigen sources for vaccines.The genetic modification of Leishmania species described in this research is a promising vaccine platform for developing vaccines for future generations.

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